ID #1124 Hijacking Neurodevelopmental Fate Decisions in Diffuse Midline Glioma

Abstract Diffuse midline glioma, K27-altered (DMG) is a uniformly fatal cancer of the brainstem, thalamus, and spinal cord that usually arises in children. Next-generation sequencing in the last decade has revealed that 80% of patients with DMG harbour K27M mutations in histone variants H3.1 or H3.3, leading to global loss of H3K27me3 and a halted differentiation. Cells subsequently acquire oncogenic mutations resulting in tumour cells that transcriptionally resemble OPCs. DLX2 is a homeobox transcription factor necessary for GABAergic interneuron differentiation during healthy neurogenesis, which directly represses transcription factors necessary for OPC differentiation. We hypothesised that by pushing differentiation away from the halted OPC fate it would promote differentiation and lead to a reduction in tumourigenicity. RNA-sequencing data from 60 DMG cell lines revealed that inverse expression profiles of DLX2 and OLIG2 were maintained. Therefore, to test the plasticity of DMG cell fate two H3.1K27M and H3.3K27M cell lines underwent DLX2 overexpression, with the highest proportion of DLX2 expressing cells showing activation of DLX2 target neuronal genes by qPCR. Bulk RNAseq revealed activated neuronal programs, but that the cells maintained OPC and mesenchymal gene programs, suggesting incomplete transdifferentiation. Comparison of this data to an existing scRNAseq database of the fetal human brain showed subtype specific differences in transcriptional plasticity between H3.3 and H3.1 K27M cell lines. Thus, we show that transdifferentiation and transcriptional plasticity as it pertains to the existing developmental lineages present in DMG are dictated by histone subtype. Analysis of changes in chromatin structure and DNA methylation, as well as tumourigenesis in vitro and in vivo following DLX2 overexpression will be presented. These data show that mmanipulation of cell fate determination through altering a key transcriptional regulator in DMG provides a developmentally relevant model system for assessing the contribution differentiation and tumourigenicity of DMG.